Antimicrobial Efficacy of Plasma-Activated Water Against Enterococcus faecalis Biofilms: An In Vitro and Ex Vivo Study

Abstract

Abstract This study evaluated the antimicrobial efficacy of plasma-activated water (PAW) against Enterococcus faecalis biofilms using in vitro and ex vivo tooth models. E. faecalis biofilms were cultivated in 96-well plates and exposed to PAW with activated times of 2, 4, 6, 8, 10, and 12 minutes including 2.5% sodium hypochlorite (NaOCl). After contact times of 5 minutes, colony-forming units were counted, and percentage of bacterial inhibition were calculated to assess the antimicrobial efficacy. Thirty extracted single-rooted teeth were decoronated, instrumented, and inoculated with E. faecalis for 7 days to allow biofilm formation within the root canals. Specimens were randomly allocated into three groups (n = 10) based on the root canal irrigants used: 0-minute PAW (negative control), 8-minute PAW, and 2.5% NaOCl (positive control). All canals were irrigated with 20 mL of respective solutions for 5 minutes. After the irrigation protocol, the samples were sectioned longitudinally and examined using scanning electron microscopy (SEM) to assess biofilm structure. Data were analyzed using the Kruskal–Wallis test followed by Dunn's post hoc comparisons. In vitro study showed a significant increase in percentage of bacterial inhibition with increasing plasma activation time, with PAW activated for 8, 10, and 12 minutes demonstrating no significant difference compared to 2.5% NaOCl (p > 0.05). Irrigation of the root canals with 8-minute PAW resulted in significantly greater bacterial reduction compared to the negative control, with no statistically significant difference in percentage of inhibition compared to 2.5% NaOCl (p = 0.175). SEM analysis demonstrated disruption of the biofilm architecture following irrigation with 8-minute PAW. PAW demonstrated antimicrobial efficacy against E. faecalis biofilms under the tested conditions. However, as this study examined a monospecies biofilm in ex vivo tooth models, whereas endodontic infections typically involve polymicrobial biofilms, PAW may be more effective when used as an adjunctive irrigant for root canal disinfection.